JP6779616B2 - NKT cell activating pharmaceutical composition, method for producing the same, and method for storing antigen-presenting cells. - Google Patents

Description

The present invention relates to an NKT cell activating pharmaceutical composition, a method for producing the same, and a method for preserving antigen-presenting cells.

As an effective treatment method for malignant tumors and the like, immunotherapy that activates natural killer T (NKT) cells in the subject body is known. NKT cells have antitumor activity. It is known that NKT cells are proliferated and activated by α-galactosylceramide (αGalCer). As a method for activating NKT cells, a method of administering a cell preparation containing dendritic cells (antigen presenting cells) derived from a subject presented with αGalCer to a subject is used (Non-Patent Document 1).

In such cell preparations, the antigen-presenting cells contained therein can be stored for a short period of time. On the other hand, depending on the condition of the subject, it may be desirable to delay the timing of use of the cell preparation. However, once the preparation is started, the administration date cannot be changed significantly depending on the condition of the subject. In addition, it cannot be transported because the storage period is short. Therefore, the cell preparation is cultivated and prepared in a facility where the subject is administered so that the antigen-presenting cells contained therein can be administered in a highly viable state, and then as much as possible. It is being administered to the subject promptly.

Ishikawa E.I. et al. , Int. J. Cancer, 117,265-273 (2005)

An object of the present invention is to provide an NKT cell-activating pharmaceutical composition having an improved storage period, a method for producing the same, and a method for storing antigen-presenting cells.

The NKT cell activating pharmaceutical composition according to the embodiment expresses a CD1d molecule on its surface, and α-galactosylceramide is bound to the CD1d molecule, with respect to the dendritic cell as an active ingredient and the total volume. 40 volumes /% by volume or more of plasma is contained in the physiological solution.

According to an embodiment of the present invention, it is possible to improve the shelf life of antigen-presenting cells in a pharmaceutical composition for activating NKT cells.

It is a schematic diagram which shows an example of the antigen presenting cell of an embodiment. It is a flowchart which shows the example of the manufacturing method of the pharmaceutical composition of an embodiment. It is a flowchart which shows the example of the manufacturing method of the pharmaceutical composition of an embodiment. It is a flowchart which shows the example of the preservation method of the antigen-presenting cell of an embodiment. It is a figure which shows the result in Example 1 of Embodiment. It is a graph which shows the result of Example 7 of Embodiment.

Hereinafter, embodiments will be described with reference to the drawings.

1. 1. Pharmaceutical Composition for Activating NKT Cells An example of a pharmaceutical composition according to an embodiment expresses a CD1d molecule on its surface as an active ingredient, and the CD1d molecule is referred to as α-galactosylceramide (hereinafter referred to as “αGalCer”). ) Is bound to the dendritic cells, and plasma having a concentration of 40 volumes / volume% or more based on the total volume is contained in the physiological solution. A dendritic cell that expresses a CD1d molecule on its surface and has αGalCer bound to the CD1d molecule is hereinafter referred to as an antigen-presenting cell.

Antigen-presenting cells present αGalCer via a CD1d molecule expressed on monocyte-derived dendritic cells. With such a structure, the antigen-presenting cell directly activates the NKT cell by presenting αGalCer as a ligand in the body of the subject to which it is administered. When αGalCer binds to the receptor of NKT cells, NKT cells are activated, and the activated NKT cells directly attack the tumor cells and at the same time cause the production of IFN-γ and the like. The resulting IFN-γ activates both NK cells and CD8 T cells, each of which indirectly attacks malignant tumor cells. In addition, dendritic cells carrying αGalCer mature by stimulation of CD40L from activated NKT cells, and as a result, activate CD8T cells. By such a mechanism of action, the pharmaceutical composition according to the embodiment is effective in the treatment of malignant tumors.

Examples of malignant tumors are skin cancer, lung cancer, thyroid cancer, gastric cancer, esophageal cancer, bile duct cancer, colon cancer, rectal cancer, liver cancer, renal cancer, pancreatic cancer, head and neck cancer, breast cancer, bladder cancer, ovarian cancer, uterine cancer, It can be a prostate cancer, a testis tumor, a childhood cancer, a malignant lymphoma, a myeloma, a brain tumor, a leukemia, a bone / soft tumor, or a combination thereof.

The subject can be, for example, any mammal such as rodents such as mice and rats, companion animals such as rabbits, dogs and cats, domestic animals such as horses and cows, and primates such as monkeys and humans.

In the prior art, it is common to suspend antigen-presenting cells in physiological saline supplemented with albumin and administer them to a subject. In such a suspension, it is very difficult to secure a viability of 45% or more of antigen-presenting cells for 3 days or more. According to the NKT cell activating pharmaceutical composition according to the embodiment, the antigen-presenting cells can be stored for a longer period than before, for example, about 8 days after production, while maintaining a survival rate of 45% or more. It is also possible to transport the pharmaceutical composition from the place of manufacture to the place of administration to the subject within about 24 hours.

2. Ingredients contained in a pharmaceutical composition for activating NKT cells (1) Antigen-presenting cells The pharmaceutical composition according to the embodiment is a composition for activating NKT cells and contains antigen-presenting cells as an active ingredient. .. As shown in FIG. 1, the antigen-presenting cell 1 refers to a cell in which αGalCer4 is bound to a CD1d molecule 3 on the surface of a dendritic cell 2.

The dendritic cell can be a dendritic cell expressing a CD1d molecule on its surface. It may be an in vitro differentiated dendritic cell or an in vivo differentiated dendritic cell. Dendritic cells differentiated in vitro can be dendritic cells of mononuclear origin. The mononucleosis is, for example, a peripheral blood mononucleosis. The dendritic cells differentiated in vivo can be, for example, myeloid dendritic cells collected from a subject, tissue-derived dendritic cells, or a combination thereof. For example, antigen presenting cells may be present at a concentration of in the pharmaceutical composition 1.0 × 10 ⁷ cells /ML～2.5×10 ⁷ cells / mL.

(2) Physiological solution In a pharmaceutical composition for activating NKT cells, antigen-presenting cells are contained in a physiological solution. The physiological solution is a carrier for maintaining the components contained in the pharmaceutical composition and carrying the components contained therein into a living body. Examples of physiological solutions can be saline, physiological buffers, electrolytes, sugar solutions and the like. The physiological solution preferably has a pH value of 3.5 to 8.0 and is not biotoxic or irritating.

The physiological solution may contain antigen-presenting cells as an active ingredient and plasma having a concentration of 40% by volume / volume% or more based on the total volume as an ingredient for storing the antigen-presenting cells. Plasma is about 40 volumes / volume% or more, about 45 volumes / volume% or more, for example, 40 volumes / volume% to 60 volumes / volume%, 45 volumes / volume% to the total volume in the pharmaceutical composition. It may be included in 55 volumes /% by volume, or 45 volumes /% by volume to 60% by volume / volume. The inclusion of plasma in such concentrations in physiological solutions maintains high viability of antigen-presenting cells and improves shelf life.

Therefore, a physiological solution containing plasma having a concentration of 40% by volume / volume% or more is a carrier of the components contained in the pharmaceutical composition, and at the same time, the antigen-presenting cells can be stored for a long period of time. It also plays the role of a preservation solution. Hereinafter, a physiological solution containing plasma having a concentration of 40% by volume / volume% or more is referred to as a "preservative solution".

The plasma may be autologous plasma or allogeneic plasma. Autologous plasma is plasma from the subject to which the pharmaceutical composition is administered. The allogeneic plasma can be plasma from a mammal other than the subject to which the pharmaceutical composition is administered.

(3) Other components for preservation The preservation solution is a component for storing antigen-presenting cells, and is a cytokine (hereinafter referred to as "cytokine") that signals through a receptor containing a common gamma chain. , And / or may include insulin. Further, the pharmaceutical composition may contain albumin as a component that stably retains the protein in the preservation solution.

Cytokines are characterized in that their receptors contain a common gamma chain (Common Gamma Chain, γc, CD132). The receptor may contain an α chain, a β chain, or the like in addition to the gamma chain. The receptor can be, for example, an IL-2 receptor, an IL-7 receptor, an IL-15 receptor, and the like. Cytokines make it possible to maintain high viability of antigen-presenting cells for extended periods of time. Cytokines can be, for example, IL-2, IL-7, IL-15 and the like. These can be used alone or in combination. The concentration of the cytokine in the pharmaceutical composition can be, for example, 1 JRU / mL to 1000 JRU / mL.

Insulin is one of the growth factors and is a growth factor capable of stabilizing and maintaining the state of antigen-presenting cells. A growth factor having such characteristics can be used not only with insulin but also with other types of components in place of insulin, in part, or in combination with insulin. The concentration of insulin in the pharmaceutical composition can range from 0.0142 U / mL to 0.142 U / mL.

The other type of component can be, for example, an insulin analog having a range of mutations capable of stabilizing and maintaining the state of antigen-presenting cells.

Albumin can be human albumin when the pharmaceutical composition is administered to humans. When the pharmaceutical composition is administered to a mammal other than human, it can be selected according to the animal species of the mammal. The concentration of albumin in the pharmaceutical composition can be 5% -10%.

(4) Other components In addition to the components described in (2) to (4), the pharmaceutical composition for activating NKT cells includes lysis aids, buffers, etc. that do not affect the activity of each component. It may include isotonic agents, stabilizers, soothing agents, or a combination thereof.

The NKT cell activating pharmaceutical composition prepared as described above can be administered to a subject and used. The route of administration may be, for example, intradermal, subcutaneous, intramuscular, intraarterial, intravenous, intratumoral, intramucosal, submucosal, intraperitoneal, or a combination thereof. The dosage form can be, for example, injection or infusion.

The NKT cell-activating pharmaceutical composition can be administered to the subject in effective amounts at one time. Here, the effective amount can be selected depending on the age, height, body weight, sex, degree of progression of malignant tumor, etc. of the subject to which the NKT cell activating pharmaceutical composition is administered.

Further, when mononuclear cells are divided into two and cultured as shown in FIG. 4 and the first and second NKT cell activating pharmaceutical compositions are administered, the first NKT cell activating pharmaceutical composition is administered to the subject. It is preferable that the second NKT cell activating pharmaceutical composition is administered to the subject 3 to 7 days after the culturing.

3. 3. Method for Producing NKT Cell-Activating Pharmaceutical Composition According to the embodiment, a method for producing a pharmaceutical composition for activating NKT cells is provided. The manufacturing method may include the following steps (a) to (c) as shown in FIG.
(A) Differentiate mononuclear cells to obtain dendritic cells expressing CD1d molecules on the surface.
(B) Bind αGalCer to the dendritic cells obtained in (a) to obtain antigen-presenting cells, and (c) 40 volumes / volume of the antigen-presenting cells obtained in (b) with respect to the total volume. To obtain an NKT cell-activating pharmaceutical composition by containing it in a preservation solution containing the plasma having a concentration of% by volume or more.

The method for producing such an NKT cell-activating pharmaceutical composition will be further described below.

(A) Differentiation of monocytes into dendritic cells expressing CD1d molecules on the surface Differentiation of monocytes into dendritic cells expressing CD1d molecules on the surface is, for example, converting monocytes into mononuclear cells. This can be done by culturing in a medium containing components that differentiate into dendritic cells. The medium may contain, for example, the cytokine, granulocyte macrophage colony stimulating factor (GM-CSF) and plasma. Cytokines, GM-CSF and plasma can be contained in the medium at concentrations of, for example, about 100 JRU / mL, about 800 U / mL, and about 2.5%, respectively.

The basal medium of the medium may be a liquid medium containing the above-mentioned members and capable of culturing cells, and may be, for example, AIM-V (registered trademark) or RPMI-1640.

The medium may be a commercially available medium, for example, X-VIVO (registered trademark) (LONZA), Humankine G4 Dendritic Cell Generation Kit (Humanzyme), or dendritic cell differentiation medium (Takara Bio). And so on.

When culturing in the medium as described above, the cells expressing the CD1d molecule on the cell surface in the mononuclear cells differentiate into dendritic cells about 5 to 7 days after the start of culturing. obtain. The differentiation can be induced by CD11c ^- CD3 ⁺ T cells contained in the mononuclear cells.

(B) Preparation of Antigen-Presenting Cells For the antigen-presenting cells, for example, αGalCer is added to the dendritic cells obtained by culturing the mononuclear cells in the medium for 6 to 13 days, and then for another 1 to 2 days. It can be obtained by culturing. Here, the addition of αGalCer may be the addition of an αGalCer solution prepared so that the concentration of αGalCer in the medium is 100 to 200 ng / mL. The αGalCer solution can be prepared, for example, by dissolving αGalCer in a solvent such as distilled water. αGalCer can be, for example, KRN7000.

In a further embodiment, the antigen presenting cells can be prepared by a method comprising the following steps shown in FIG.

Dividing the mononuclear cells into two and differentiating the first mononuclear cells into first dendritic cells,
The second mononuclear cells were cultured in a medium similar to the medium used for culturing the first mononuclear cells for a longer period than that of the first peripheral blood cells, for example, 10 to 14 days, to become a second dendritic cell. Differentiation, and αGalCer is added to the first dendritic cell and the second dendritic cell, respectively, and cultured for 1 to 2 days to obtain a first antigen-presenting cell and a second antigen-presenting cell. ..

The first mononuclear cells and the second mononuclear cells can each differentiate into dendritic cells at the same time, and the second dendritic cells can be cultured as dendritic cells for a longer period of time. When the first antigen-presenting cell and the second antigen-presenting cell have been cultured for a predetermined period of time, they are similarly collected by the method described later, suspended in separate storage solutions, and separately. Can be housed and stored in a container.

(C) Mixing of antigen-presenting cells into a storage solution and preparation of an NKT cell-activating pharmaceutical composition The antigen-presenting cells prepared as described above can be recovered when the culture for a predetermined period is completed. Recovery can be performed, for example, by pipetting antigen-presenting cells into a centrifuge tube and then centrifuging. Centrifugation is preferably carried out at 1200 rpm to 1500 rpm for 5 to 10 minutes at room temperature. The recovered antigen-presenting cells may be washed with human albumin-added physiological saline. The number of recovered antigen-presenting cells can then be measured. The number of cells may be about 2 × 10 ⁹ to about 5 × 10 ⁹ . The cell number may be measured by any known method.

The storage solution includes physiological saline as a solvent, a physiological buffer solution, an electrolyte solution, or a physiological solution such as a sugar solution, and plasma contained at a concentration of 40 volume / volume% or more based on the total volume. The storage solution can be prepared by adding 40% or more of plasma by volume to a physiological solution sterilized by filtration or high-pressure steam sterilization. The plasma may be autologous plasma or allogeneic plasma.

Plasma can be used by obtaining one prepared at a medical institution or the like, or one commercially available. In medical institutions and the like, plasma can be collected, for example, as follows. First, blood can be drawn from a donor that provides plasma. Blood preferably contains about 100 mL to 300 mL of plasma. Mononucleosis can then be removed from the blood. It is preferable that at least 80 mL to 240 mL of plasma remains in the blood from which mononuclear cells have been removed. The autologous plasma can be derived from blood taken from a subject to whom the pharmaceutical composition is administered. The allogeneic plasma can be blood collected from a mammal other than the subject to which the pharmaceutical composition is administered. The plasma can be collected from the same blood as the blood from which the monocytes are collected or from blood collected separately. The plasma can be used in steps (a) and / or (c).

In addition to the plasma, the cytokine, insulin, albumin, or any combination thereof may be added to the preservation solution. Cytokines can be, for example, IL-2, IL-7, IL-15, etc., and IL-2 can be, for example, Immunoce® (Shionogi Pharmaceuticals, Japan). Insulin can be, for example, Humarin® R Note 100 units / mL. Albumin can be, for example, blood donated albumin (Kaketsuken).

In a further embodiment, the preservation solution prepared with allogeneic plasma can be prepared with a blood product. For example, blood preparations may be used in place of plasma and albumin of 40% by volume / volume% or more of the storage solution. The blood product may be a mixture of a blood component from which the leukocyte component has been removed and a blood preservation solution. The blood preservation solution may be, for example, an ACD-A solution. The blood product can be, for example, fresh frozen plasma-LR "JRC". As the blood product, one suitable for the negative positive of the ABO blood group and D (Rho) antigen of the subject to which the NKT cell activating pharmaceutical composition is administered can be used. In that case, plasma is 2. A preservation solution can be prepared by diluting the blood product with a physiological solution so as to have the concentration described in (2). 2. Cytokines and insulin are added to the preservation solution. It may be added so as to have the concentration described in (3) of.

Antigen-presenting cells are included in the preservation solution prepared as described above to obtain an NKT cell-activating pharmaceutical composition.

The mononucleosis used in the production method may be prepared at a medical institution or the like, or may be commercially available. In a medical institution or the like, mononucleosis can be collected as follows, for example. First, blood can be drawn from a donor that provides peripheral blood cells. The collected blood preferably contains about 2 × 10 ⁹ to 10 × 10 ⁹ monocytes. Mononucleosis can then be collected from the collected blood. It can be done, for example, by diluting the blood with saline, centrifuging, and collecting a layer of monocytes from the centrifuged blood. The mononucleosis is preferably an infectious mononucleosis of the target, but may be a mononucleosis obtained from a mammal other than the target by the same method as described above. The steps (a) to (c) can be performed aseptically.

In a further embodiment, the pharmaceutical composition for activating NKT cells can be produced by the following steps.

(A) Culturing mononuclear cells in a medium containing cytokines, GM-CSF, and a part of the plasma to differentiate them into dendritic cells.
(B) Bind αGalCer to the dendritic cells obtained in (a) to obtain antigen-presenting cells.
(C) Collecting the antigen-presenting cells,
(D) Prepare a preservation solution containing cytokines, insulin, and the plasma having a volume ratio of 40% or more, and (e) 40 volumes / volume of the antigen-presenting cells obtained in (d) with respect to the total volume. To obtain an NKT cell-activating pharmaceutical composition by containing it in a preservation solution containing the plasma having a concentration of% or more.

The steps (a) to (e) may be the same as the above-mentioned manufacturing method.

4. Method for Preserving NKT Cell-Activating Pharmaceutical Composition The method for preserving the above-mentioned NKT cell-activating pharmaceutical composition is described below. According to embodiments, a method of preserving the pharmaceutical composition for activating NKT cells is provided. As shown in FIG. 4, the preservation method may include the following steps (a) to (d). (A) Differentiate mononuclear cells to obtain dendritic cells expressing a CD1d molecule.
(B) To obtain antigen-presenting cells by binding αGalCer to the dendritic cells obtained in (a) above.
(C) To prepare an NKT cell-activating pharmaceutical composition by incorporating the antigen-presenting cells obtained in (b) above into a preservation solution containing plasma having a concentration of 40 volumes / volume% or more based on the total volume. And (d) The NKT cell-activating pharmaceutical composition obtained in (c) above is maintained at a temperature (T) of 0 ° C. <T ≤ 8 ° C.

The steps up to obtaining the NKT cell-activating pharmaceutical composition according to (a) to (c) may be the same as the above-mentioned production method.

The NKT cell-activating pharmaceutical composition prepared as described in (a) to (c) can be contained in a container in a state suitable for storage and transportation. The container may be, for example, a drip bag, ampoule, syringe, tube, or pack. Containment is performed aseptically and can be sealed after containment of the composition. Then, it is brought into a cold storage set at a temperature (T) of 0 ° C. <T ≦ 8 ° C. and maintained. The temperature of the cold storage is more preferably 2 ° C to 6 ° C, and may be, for example, about 4 ° C. Maintenance of the process can be performed in a sealed state. Thereby, the antigen-presenting cells in the NKT cell-activated pharmaceutical composition can be preserved without gas exchange and preservation solution exchange.

When the NKT cell activating pharmaceutical composition is produced and stored as described above, the viability of the antigen-presenting cells is maintained at 45% or more for about 8 days. Transportation is possible within 24 hours out of 8 days. For transport, the NKT cell-activating pharmaceutical composition may be maintained at 0 ° C to 8 ° C during transport. The NKT cell-activating pharmaceutical composition can be transported in a shaded environment.

In a further embodiment, the antigen-presenting cells in the pharmaceutical composition for activating NKT cells can be preserved by the following steps.

(A) Culturing mononuclear cells in a medium containing cytokines, GM-CSF, and a part of the plasma to differentiate them into dendritic cells.
(B) Bind αGalCer to the dendritic cells obtained in (a) to obtain antigen-presenting cells.
(C) Collecting the antigen-presenting cells,
(D) To prepare a preservation solution containing cytokines, insulin, and the plasma having a volume ratio of 40% or more.
(E) The antigen-presenting cells obtained in (d) are included in a preservation solution containing plasma at a concentration of 40 volumes / volume% or more based on the total volume to obtain an NKT cell-activating pharmaceutical composition, and ( f) The NKT cell activating pharmaceutical composition obtained in (c) is allowed to stand in a cold storage at a temperature (T) of 0 ° C <T ≤ 8 ° C.

The steps (a) to (f) may be the same as the above-mentioned storage method.

<Example>
Hereinafter, examples of the method for producing the NKT cell-activating pharmaceutical composition and the method for preserving antigen-presenting cells described in the embodiments will be described.

Example 1. Characteristic evaluation of peripheral blood mononuclear cells cultured in IL-2 / GM-CSF Peripheral blood mononuclear cells collected from the subjects HV1 and HV2 were centrifuged in a density gradient, washed once with PBS, and 3% heat-inactivated. It was washed twice with PBS or RPMI-1640 supplemented with FBS. Washed peripheral blood mononuclear cells containing 100 JRU / mL rhIL-2 (Immnes®, Shionogi Pharmaceutical) and 800 U / mL rhGM-CSF (NCPC Genetech Biotechnology Development), 10% FBS , 0.01 m M2-ME, and RPMI-1640 supplemented with 50 U / mL penicillin-streptomycin for 7 days. The obtained IL-2 / GM-CSF cultured peripheral blood mononuclear cells were cultured in the presence of 100 JRU / ml IL-2 and 800 U / ml GM-CSF, and the cells were cultured by flow cytometry (FCM). The size and expression of CD11c and HLA-DR were analyzed. IL-2 / GM-CSF cultured peripheral blood mononuclear cell SSC / FSC plots before and after culture are shown in the upper part of A in FIG. 5, and HLA-DR / CD11c plots of cells are shown in the lower part of A in FIG. .. The peripheral blood mononuclear cells contained 15% SSC ^high FSC ^high cells, in which 23.8% dendritic cells (HLA-DR ⁺ CD11c ⁺ cells) were contained. In addition, FCM using anti-CD11c-PE and anti-CD3-Cy (Pharmingen) revealed that the peripheral blood mononuclear cells also contained CD11c ^- CD3 ⁺ cells.

Example 2. Evaluation of survival rate of antigen-presenting cells stored in a preservation solution containing no IL-2, insulin, and autologous plasma Peripheral blood mononuclear cells from blood collected from two healthy 31-year-old males and two healthy 34-year-old males. Cells are harvested and cultured with 100 JRU / mL IL-2 and 800 U / mL rhGM-CSF for 6 or 13 days, αGalCer (100 ng / mL, KRN7000) added, at 37 ° C. and 5% CO ₂ conditions. It was cultured for 1 day. αGalCer is described by Kawano et al. , Science, 1997. In this way, antigen-presenting cells cultured for a total of 7 days (7-day cultured cells) and antigen-presenting cells cultured for a total of 14 days (14-day cultured cells) were prepared. After collecting a part of each antigen-presenting cell and staining it with trypan blue, the number of cells was measured and it was confirmed that 90% or more of the living cells were present. The remaining cells were collected and dispensed and suspended in 9 tubes containing 25 mg / mL albumin-containing saline to a concentration of 1.5 × 10 ⁶ cells / 100 μL. After storing each tube under the conditions of 4 ° C., 10 ° C., or 22 ° C. for 24 hours, 48 hours, or 72 hours under a total of 9 conditions, the cell viability was calculated. The average value of the three samples was calculated.

The results are shown in Table 1. In both the 7-day cultured cells and the 14-day cultured cells, the survival rate of 45% or more was maintained even after 72 hours under the conditions of 10 ° C. and 22 ° C., but not under the conditions of 4 ° C. Therefore, it was clarified that a preservation solution containing no IL-2, insulin, and autologous plasma cannot be stably preserved for 3 days or more.

Example 3. Evaluation of Survival Rate of Antigen-Presenting Cells Transported and Preserved in Preservation Solution Not Containing IL-2, Insulin, and Autologous Plasma 7-day cultured cells originating from a healthy 35-year-old male in the same manner as in Example 2. And 14 days cultured cells were prepared. Each antigen-presenting cells were harvested, so that 1.5 × 10 ⁸ cells / mL, respectively, were suspended in physiological saline containing 25 mg / mL albumin. They were split in half and placed in a 15 mL conical tube. After confirming that the cell viability is 90% or more, the cells are packed in a box at low temperature (2 ° C to 8 ° C) and room temperature (15 ° C to 25 ° C) together with a temperature logger, and at low temperature or room temperature conditions. Transported for 16 hours. The transportation was carried out by a transportation company (Celuto Co., Ltd.) from Chiba University (1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba) to the Compound Safety Research Institute (363-24, Shinei, Kiyota-ku, Sapporo-shi, Hokkaido). After that, the cells were stored at the same temperature as the transported temperature, and the number of viable cells was measured after 48 hours or 72 hours, respectively. The temperature during transport was 5.2 ° C to 5.8 ° C (low temperature) for 7-day cultured cells, 19.4 ° C to 22.2 ° C (room temperature), and 4.4 ° C to 5.4 for 14-day cultured cells. The temperature was 19.6 ° C. to 21.9 ° C. (room temperature).

As a result, the survival rate was 45% or more until 72 hours later only under the low temperature condition of the 7-day cultured cells (Table 2). Therefore, it was clarified that the storage temperature is preferably 2 ° C to 8 ° C.

Example 4. Evaluation of Survival Rate of Antigen-Presenting Cells Stored in the Preservative Solution of the Example 7-day and 14-day cultured cells of human origin of a healthy 47-year-old man were prepared by the same method as in Example 2. Each cell contains 25 mg / mL of albumin, 100 U / mL of IL-2, 5 μg / mL of insulin, and 10, 20, 30, 40, or 50% of autologous plasma in IL-2 and high insulin concentration 5 Conditions (high), or IL-2 and insulin concentrations containing 25 mg / mL albumin, 10 U / mL IL-2, 0.5 μg / mL insulin, and 10, 20, 30, 40 or 50% autologous plasma. It was suspended in a storage solution under a total of 10 conditions under 5 low conditions (low). Albumin is 25% blood donated albumin intravenous injection 5g / 50mL "Benesis" (Tanabe Mitsubishi Pharmaceutical Co., Ltd.), IL-2 is Imnes (registered trademark) Note 35 (Shionogi Pharmaceutical), insulin is Humarin (registered trademark) R (Nippon Eli Lilly Co., Ltd.) was used. They were stored at 4 ° C. and cell viability was measured after 96 hours, 144 hours, 240 hours and 336 hours.

The results are shown in Table 3. It was revealed that cells can be preserved with a survival rate of 45% for 6 days in a preservation solution containing IL-2, insulin, and 40% or more plasma. It was also clarified that the higher the concentration of IL-2 and insulin, the higher the proportion of living cells, but if the plasma concentration is 40% or more, it can be stored for 6 days.

Example 5. Evaluation of viability of antigen-presenting cells stored in the preservation solution of the embodiment 7-day cultured cells of human origin of a healthy 47-year-old man and 7-day cultured cells of human origin of a healthy 35-year-old man by the same method as in Example 2. And 14 days cultured cells were prepared. Each antigen-presenting cell contains 25 mg / mL of albumin, 100 U / mL of IL-2, 5 μg / mL of insulin, and IL-2 containing 50% of autologous plasma, and high insulin concentration (high), or 25 mg of insulin. Storage of IL-2 containing / mL, IL-2 at 10 U / mL, insulin at 0.5 μg / mL, and autologous plasma at 40, 45 or 50%, and 3 conditions with low insulin concentration (low), for a total of 4 conditions Suspended in liquid. For albumin, IL-2 and insulin, the same products as in Example 4 were used. After storing them at 4 ° C. for 96 hours, 144 hours or 192 hours, the viability of the cells was measured, respectively.

Table 4 shows the results in humans of 47-year-old men, and Table 5 shows the results in humans of 35-year-old men. Under the experimental conditions of the 7-day cultured cells, the IL-2 and insulin concentrations were low in the case of a 47-year-old male human, and the survival rate was maintained at 45% or more for 8 days under the condition of containing 40% plasma. In the case of a 35-year-old man, the survival rate was maintained at 45% or more for 8 days under the condition that the plasma content was 45% or more. 14-day cultured cells were maintained for 6 days under conditions of high IL-2 and insulin concentrations and 50% plasma. Therefore, it was clarified that the 7-day cultured cells can be stored for a maximum of 8 days and the 14-day cultured cells for a maximum of 6 days with the preservation solution of the embodiment.

Example 6. Evaluation of Survival Rate of Antigen-Presenting Cells Derived from Blood Transported or Not Transported in the Preservative Solution of the Embodiment Blood was collected from a healthy 35-year-old male human and one of the blood was collected at Chiba University in the same manner as in Example 2. 7-day cultured cells and 14-day cultured cells were prepared from the department. The rest of the blood was transported to the Regenerative Medicine Research Department of FUJISOFT Co., Ltd. (2-19-7 Kotobashi, Sumida-ku, Tokyo), and cultured cells on the 7th and 14th were prepared in the same manner. The transfer was performed by dispensing the blood into tubes and packing them in a box at 15 ° C to 25 ° C with a temperature logger. The transport temperature was 15 ° C to 25 ° C. In each of the above-mentioned locations, each antigen-presenting cell contains 25 mg / mL of albumin, 100 U / mL of IL-2, 5 μg / mL of insulin, and 50% of autologous plasma in IL-2 and high insulin concentration (high). ), Or albumin 25 mg / mL, IL-2 10 U / mL, insulin 0.5 μg / mL, and IL-2 containing 40, 45, or 50% autologous plasma, and low insulin concentration 3 conditions (low) ) Was suspended in a storage solution under a total of 4 conditions. For albumin, IL-2 and insulin, the same products as in Example 4 were used. After storing them at 4 ° C. for 4 days, the cells were packed and transported in a box at 2 ° C. to 8 ° C. with a temperature logger. The transportation was carried out by a transportation company (Celuto Co., Ltd.) from Chiba University or FUJISOFT Co., Ltd. Regenerative Medicine Research Department to the Compound Safety Research Institute. The transport time was about 24 hours. The transport temperature was 2 ° C to 8 ° C. The number of cells was measured 24 hours, 48 hours, and 72 hours after the transfer.

The results are shown in Table 6. Survival rate of 45% or more is maintained for 72 hours after transport under conditions other than the condition in which 14-day cultured cells of blood origin that have not been transported are stored in a preservation solution containing IL-2 and high insulin concentration and 50% plasma. It was. Therefore, it was clarified that the cells were preserved for up to 8 days even if they were transported for 24 hours during the storage period.

Example 7. NKT cell activation efficiency test of antigen-presenting cells stored in the preservation solution of the embodiment 7-day cultured cells of human origin of a 35-year-old man prepared in Example 6 are the same as in Example 6 (high) or Example 6 (low). The cells were suspended in a preservation solution of 5 conditions in total, or a preservation solution containing only albumin (25 mg / mL). Cells cultured for 7 days without the addition of αGalCer to peripheral blood mononuclear cells of the same subject origin as controls were suspended in the above (high) preservation solution. These were stored at 4 ° C. for 4 days and then irradiated to kill NKT cells. Peripheral blood mononuclear cells were newly collected from the same subject, Vα24 and Vβ11 were detected using FACS, and the number of activated NKT cells was measured (FIG. 7 “at the start of culture”). Irradiated cultured cells and fresh peripheral blood mononuclear cells were co-cultured for 7 days, and the number of activated NKT cells was measured.

The results are shown in FIG. In any of the conditions, the number of activated NKT cells was larger in the condition where αGalCer was added than in the case where αGalCer was not added. Therefore, it was revealed that the cells stored in the preservation solution of the embodiment activate NKT cells.

From Examples 2 to 7, the pharmaceutical composition of the embodiment can store antigen-presenting cells at a survival rate of 45% or more for up to 8 days, and maintains a survival rate of 45% or more even after transport within 24 hours. It was revealed that the pharmaceutical composition still activates NKT cells even after storage.
The inventions described in the claims at the time of filing of the present application are described below.
[1]
A dendritic cell as an active ingredient, which expresses a CD1d molecule on its surface and has α-galactosylceramide bound to the CD1d molecule, and
Plasma at a concentration of 40 volumes / volume% or more based on the total volume
A pharmaceutical composition comprising the above in a physiological solution.
[2]
The pharmaceutical composition according to [1], wherein the plasma concentration is 40 to 60 volumes / volume%.
[3]
The pharmaceutical composition according to [1], wherein the plasma concentration is 45 to 55 volumes / volume% or more.
[4]
The pharmaceutical composition according to [1], wherein the plasma concentration is 45% by volume / volume% or more.
[5]
The pharmaceutical composition according to any one of [1 to 4], further comprising a cytokine that signals via a receptor comprising a common gamma chain.
[6]
The pharmaceutical composition according to any one of [1] to [5], further comprising insulin or an insulin analog.
[7]
The pharmaceutical composition according to any one of [1] to [6], further comprising albumin.
[8]
The pharmaceutical composition according to any one of [1] to [7], wherein the plasma is autologous plasma.
[9]
The pharmaceutical composition according to any one of [1] to [8], wherein the plasma is allogeneic plasma.
[10]
(A) Differentiate mononuclear cells to obtain dendritic cells expressing the CD1d molecule.
(B) Bind α-galactosylceramide to the dendritic cells obtained in (a) above to obtain antigen-presenting cells, and
(C) To prepare an NKT cell-activating pharmaceutical composition by incorporating the antigen-presenting cells obtained in (b) above into a preservation solution containing plasma at a concentration of 40 volumes / volume% or more based on the total volume.
A method for producing a pharmaceutical composition for activating NKT cells containing.
[11]
(A) Differentiate mononuclear cells to obtain dendritic cells expressing the CD1d molecule.
(B) To obtain antigen-presenting cells by binding α-galactosylceramide to the dendritic cells obtained in (a) above.
(C) To prepare an NKT cell-activating pharmaceutical composition by incorporating the antigen-presenting cells obtained in (b) above into a preservation solution containing plasma at a concentration of 40 volumes / volume% or more based on the total volume. ,as well as
(D) The NKT cell-activating pharmaceutical composition obtained in (c) above is maintained at a temperature (T) of 0 ° C. <T ≤ 8 ° C.
A method of preserving antigen-presenting cells containing.
[12]
The method according to [11], wherein the maintenance in (d) is carried out for any period up to 8 days.
[13]
The method according to [11] or [12], wherein the maintenance in (d) is performed in a sealed state.

1 ... Antigen-presenting cell 2 ... Dendritic cell 3 ... CD1d molecule 4 ... αGalCer.

Claims (6)

Plasma with a concentration of 40 volumes / volume% or more based on the total volume,
IL-2 and
Insulin is contained in a physiological solution,
A preservative solution for storing dendritic cells expressing a CD1d molecule on the surface and having α-galactosylceramide bound to the CD1d molecule at a temperature exceeding 0 ° C. without lowering the temperature below 0 ° C. The preservative solution according to claim 1 and
A pharmaceutical composition comprising a dendritic cell as an active ingredient, which expresses a CD1d molecule on its surface and has α-galactosylceramide bound to the CD1d molecule. The method for producing a pharmaceutical composition according to claim 2 .
(A ) Obtaining dendritic cells expressing the CD1d molecule,
(B) The dendritic cells obtained in (a) above are bound to α-galactosylceramide to obtain antigen-presenting cells, and (c) the antigen-presenting cells obtained in (b) above are added to the total volume. NKT cells comprising preparing an NKT cell-activating pharmaceutical composition by including plasma having a concentration of 40% by volume /% or more, IL-2 , and insulin in a preservation solution contained in a physiological solution. A method for producing a pharmaceutical composition for activation. (A ) Obtaining dendritic cells expressing the CD1d molecule,
(B) To obtain antigen-presenting cells by binding α-galactosylceramide to the dendritic cells obtained in (a) above.
(C) The antigen-presenting cells obtained in (b) above are contained in a preservation solution containing plasma having a concentration of 40 volumes / volume% or more based on the total volume, IL-2 , and insulin in a physiological solution. To prepare an NKT cell-activating pharmaceutical composition by containing it in (d), and (d) without lowering the NKT cell-activating pharmaceutical composition obtained in (c) above to 0 ° C. or lower.
A method for preserving antigen-presenting cells, comprising maintaining at a temperature (T) of 0 ° C <T ≤ 8 ° C. Claim 4 may be maintained in (d) is carried out over any period up to 8 days
The method described in. The method according to claim 4 or 5 , wherein the maintenance in (d) is performed in a sealed state.

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